PROJECT SUMMARY Flaviviruses that are transmitted by mosquitoes, including dengue (DV) and West Nile (WNV) viruses as well as the recently emerging Zika virus (ZIKV), represent a significant global health concern. Despite the high morbidity and mortality resulting from infection by these viral pathogens, there are currently no FDA-approved therapies. Hence, there is a pressing need to understand better the pathogenesis of these viruses to aid the design of vaccines and antivirals. Disease severity and pathogenesis of viral infections in humans depend on many factors, including pre-existing immunity, strain virulence, host genetics and virus-host interactions. Among the virus-host interactions that modulate pathogenesis, virus-mediated suppression of innate immune signaling pathways has a critical role. However, the precise mechanisms by which flaviviruses evade host innate immunity are not well characterized. The proposed study builds on a recent discovery by the Gack laboratory that the NS3 protein of DV interacts with the mitochondrial-targeting trafficking protein 14-3-3? to block the cytosol-to- mitochondria translocation of the viral RNA sensor RIG-I, thereby suppressing antiviral signaling and type I interferon (IFN) induction. We have identified the precise motif in DV NS3 for 14-3-3? interaction, a four-amino-acid phosphomimetic 64RxEP67 motif, and also generated a recombinant mutant DV. This recombinant DV, encoding a 14-3-3?-binding-deficient mutant NS3 protein, is growth-attenuated compared to wild-type DV in cells with intact innate host defense and elicits robust innate immune and T cell responses in vitro. WNV and ZIKV NS3 proteins encode a similar phosphomimetic motif, 64RLDP67, and our preliminary results show that WNV NS3 also targets 14-3-3? to block RIG-I-mediated innate immunity. Using molecular, biochemical and cell biological approaches combined with infection studies with recombinant NS3 mutant viruses, we will define in precise detail how DV and WNV NS3 inhibits the host IFN response. This study also will yield insight into the mechanism(s) by which ZIKV NS3 protein modulates IFN-mediated host defense responses (Aim 1). Finally, we will determine the physiological relevance of the 14-3-3?-NS3 interaction for viral pathogenesis and escape from host innate and adaptive immunity using in vitro cell culture systems and mouse models of WNV infection (Aim 2). Our studies will provide a molecular understanding of the immune escape mechanisms of DV, WNV and ZIKV, which may guide the rational design of new vaccines and antivirals.